Magnetic hierarchical catalysts of nanoparticles and enzymes forming mesoporous assemblies embedded in macroporous scaffolds, and their use with magnetically-driven reactors and in chemical reactions

ABSTRACT

A hierarchical catalyst composition comprising a continuous or particulate macroporous scaffold in which is incorporated mesoporous aggregates of magnetic nanoparticles, wherein an enzyme is embedded in mesopores of the mesoporous aggregates of magnetic nanoparticles. Methods for synthesizing the hierarchical catalyst composition are also described. Also described are processes that use the recoverable hierarchical catalyst composition for depolymerizing lignin, remediation of water contaminated with aromatic substances, polymerizing monomers by a free-radical mechanism, epoxidation of alkenes, halogenation of phenols, inhibiting growth and function of microorganisms in a solution, and carbon dioxide conversion to methanol. Further described are methods for increasing the space time yield and/or total turnover number of a liquid-phase chemical reaction that includes magnetic particles to facilitate the chemical reaction, the method comprising subjecting the chemical reaction to a plurality of magnetic fields of selected magnetic strength, relative position in the chemical reaction, and relative motion.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.61/710,110, filed Oct. 5, 2012, and U.S. Provisional Application No.61/767,477, filed Feb. 21, 2013, both of which are herein incorporatedby reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under contract to theNortheast Sun Grant Initiative at Cornell University US Department ofTransportation Assistance #DTOS59-07-G-00052. The government has certainrights in the invention.

BACKGROUND OF THE DISCLOSURE

Peroxidases (EC 1.11.1) are widely found in biological systems and forma subset of oxidoreductases that reduce hydrogen peroxide (H₂O₂) towater in order to oxidize a large variety of aromatic compounds rangingfrom phenol to aromatic amines. The reaction cycle of peroxidases isquite complex and begins with activation of heme by H₂O₂ to form thetwo-electron activated Compound I (N. C. Veitch, Phytochemistry, 2004,65, 249). Compound I is then reduced by one electron by the oxidation ofthe organic substrate leading to the formation of Compound II that isone electron above the resting state. The second reduction recovers theenzyme to its resting state to start a new cycle. Overall, for eachmolecule of hydrogen peroxide consumed, two aromatic free radicals areproduced and can readily react in secondary reactions.

Peroxidases are highly sensitive to substrate inhibition, mostly byH₂O₂, which can lead to the formation of the reversible inactivated formof the enzyme (Compound III). Their activities are also deterred byproduct inhibition. Therefore, the complex kinetics associated withperoxidase enzymes can restrict their use in many processes andbioprocesses. Increasing the activities of this and other families ofenzymes and their tolerance to different process conditions couldimprove their current use, as well as pave the way for their use in newapplications.

BRIEF SUMMARY OF THE DISCLOSURE

It has been discovered herein that bionanocatalysts (BNCs) consisting ofan enzyme, particularly a free-radical-producing (FRP) enzyme, such ashorseradish peroxidase (HRP), self-assembled with magnetic nanoparticles(MNPs) possess an enhanced enzymatic activity. In particular, it hasherein been found that the self-assembled clusters of enzyme andmagnetic nanoparticles generally possess faster turnover and lowerinhibition of the enzyme as compared with the free enzyme or themagnetic nanoparticle clusters without enzyme. It has herein furthermorebeen found that the size and magnetization of the MNPs affect theformation and ultimately the structure of the BNCs, all of which have asignificant impact on the activity of the entrapped enzymes.Particularly by virtue of their surprising resilience under variousreaction conditions, the BNCs described herein can be used as animproved enzymatic or catalytic agent where other such agents arecurrently used, and they can furthermore be used in other applicationswhere an enzyme has not yet been considered or found applicable.

The approach described herein sharply differs from classical methodsthat rely on protein conjugation on surface-modified particles bycomplex biochemistries, oftentimes at the expense of enzymaticactivities and reaction efficiencies. By the instant methodology, enzymekinetics are substantially modified only when the enzymes are in closeassociation with the MNPs, e.g., as a self-assembled cluster(agglomeration) of primary MNP crystallites and peroxidase enzyme. Theoverall activities of the resulting BNCs can advantageously be orders ofmagnitude higher than those of free enzymes or MNPs at biologicallyrelevant substrate concentration.

In one aspect, the invention is directed to a composition in which anenzyme is embedded (i.e., entrapped) in magnetic nanoparticles orclusters thereof. In particular embodiments, the composition is amesoporous clustered assembly of magnetic nanoparticles and one or acombination of enzymes, such as FRP enzymes. The mesoporous clusteredassemblies possess mesopores in which the enzyme is embedded. In otherembodiments, the foregoing cluster composition includes magneticnanoparticles that are surface-coated with a noble metal, such as gold.

In further embodiments, the foregoing mesoporous aggregates of magneticnanoparticles (BNCs) are incorporated into a macroporous scaffold toform a hierarchical catalyst assembly with first and second levels ofassembly. The macroporous scaffold may be constructed of an assemblageof micron-sized magnetic particles, or may be a continuous scaffold,which is not constructed from particles, or even a combination thereof.The result is a combination of highly macroporous and mesoporousmagnetic solids with enzyme functionalization, which is beneficial toimmobilization of any enzymes with small diffusible substrates andproducts. The overall hierarchical catalyst assembly is magnetic by atleast the presence of the BNCs.

In the case of a continuous macroporous scaffold, in a first set ofembodiments, the continuous macroporous scaffold in which the BNCs areincorporated is magnetic. The continuous macroporous scaffold can bemagnetic by either being composed of a magnetic polymer compositionand/or by having embedded therein magnetic particles not belonging tothe BNCs (e.g., magnetic nano- or micro-particles not associated withthe enzyme). In a second set of embodiments, the continuous scaffold inwhich the BNCs are incorporated is non-magnetic; nevertheless, theoverall hierarchical catalyst assembly containing the non-magneticscaffold remains magnetic by at least the presence of the BNCsincorporated therein.

The invention is also directed to a process for producing theenzyme-embedded magnetic nanoparticle compositions described above. Insome embodiments, magnetic nanoparticles or aggregates thereof are firstprepared, and the enzyme is subsequently absorbed therein. In otherembodiments, the enzyme-embedded magnetic nanoparticle composition isproduced by assembling monodispersed magnetic nanoparticles in thepresence of an enzyme, thereby embedding the enzyme in clusters of MNPsby a self-assembly mechanism.

The invention is also directed to a process for producing thehierarchical catalyst assembly, described above, in which mesoporousaggregates of magnetic nanoparticles (BNCs) are incorporated into acontinuous or particulate macroporous scaffold. In the method, BNCs arecontacted with the macroporous scaffold in solution to substantiallyembed the BNCs into macropores of the macroporous scaffold. In theparticular case of a continuous macroporous scaffold, the continuousmacroporous scaffold can be produced by a templating process thatincludes: (i) producing a composite containing a scaffold precursormaterial having embedded therein a sacrificial templating agent, and(ii) selective removal of the sacrificial templating agent to producemacropores in the continuous precursor material. In more specificembodiments, the templating process involves a solvent temptationprocess wherein a solvent, embedded in the scaffold precursor material,functions as a templating agent. The composite containing the scaffoldprecursor material embedded with solvent is cooled until the embeddedsolvent freezes to form solvent crystals, and then the frozen solvent isremoved by either evaporation or sublimation to produce macropores inthe scaffold precursor material. When the solvent is water, the solventtemplating process can be considered an ice temptation process.

In yet other aspects, the invention is directed to processes in whichthe above-described enzyme-embedded magnetic nanoparticle compositionsare used. In particular embodiments, the enzyme-embedded magneticnanoparticle compositions are directed to a process for depolymerizinglignin, a process for removing aromatic contaminants from water, aprocess for producing a polymer by polymerizing a monomer by a freeradical mechanism, a method for epoxidation of alkenes, a method forhalogenation of phenols, a method for inhibiting growth and function ofmicroorganisms in a solution, and a method for carbon dioxide conversionto methanol.

In still other aspects, the invention is directed to a method forincreasing a space time yield and/or total turnover number of aliquid-phase chemical reaction that includes magnetic particles, such asany of the BNCs or hierarchical catalyst assemblies thereof, tofacilitate the chemical reaction. In the method, the liquid-phasechemical reaction containing magnetic particles therein is subjected toa plurality of magnetic fields of selected magnetic strength, relativeposition in the liquid-phase chemical reaction, and relative motion tospatially confine the magnetic particles, wherein the magnetic strength,relative positioning, and relative motion of the plurality of magneticfields are provided by a system of electromagnets in which current flowis appropriately controlled or adjusted.

By virtue of the larger size and the mass magnetization of the overallhierarchical assembly containing BNCs incorporated into the macroporousframework, the enzyme-containing BNCs can be more easily captured by anexternal magnetic field, and thus, more easily removed from a reactionmedium. The simplified removal furthermore permits the more facilere-use of the catalysts. Another benefit of the hierarchical assembledcatalysts described herein is that the larger size helps to preserveenzymatic activities. Moreover, BNCs attached onto the surface ofmagnetic microparticles are less prone to over-aggregation whensubjected to magnetic fields that may be used to remove the BNCs orenhance the reaction.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B. Formation of the first level of assembly of a hierarchicalcatalyst assembly containing BNCs composed of mesoporous aggregates ofmagnetic nanoparticles and horseradish peroxidase (FIG. 1A), andformation of the second level of assembly by incorporating the BNCs intoa macroporous scaffold composed of ice-templated cellulose (FIG. 1B).

FIGS. 2A, 2B. Single-helix (FIG. 2A) and double-helix (FIG. 2B) magnetictrap arrangements as specifically applied to water remediation and/orfree radical polymerization of aromatic compounds.

FIG. 3. An electronic diagram for an arrangement ofcomputer-programmable controllers useful in independently controllingsix electromagnets or two arrays of three electromagnets.

FIG. 4. An exemplary microcontroller for use in the control input(“CtrInp”) components of the electronic arrangement provided in FIG. 3.

FIG. 5. Graph showing the effect of H₂O₂ on DMP oxidation velocitycatalyzed by manganese peroxidase at 2.5 nM, Au-M60 at differentconcentrations, and BNCs thereof at pH 3.5 in malonate buffer (50 mM).The x-axis is on a log₁₀ scale for convenience. The BNCs (gold coatednanoparticles) increase the activity and lower the inhibition ofmanganese peroxidase.

FIG. 6. Graph showing the effect of H₂O₂ on DMP oxidation velocitycatalyzed by versatile peroxidase at 2.5 nM, Au-M60 at differentconcentrations, and BNCs thereof at pH 3.5 in malonate buffer (50 mM).The x-axis is on a log₁₀ scale for convenience. The BNCs (gold coatednanoparticles) increase the activity and lower the inhibition ofversatile peroxidase

FIGS. 7A, 7B. Spectra showing use of manganese peroxidase and versatileperoxidase for lignin depolymerization of biomass feedstock (mixedgrasses). FIG. 7A shows dilution corrected spectra characteristic towater soluble lignin monomers and oligomers. FIG. 7B shows biomasscontrol corrected spectra. BNCs were formed with 25 mM of MnP and VeP(50 mM total enzyme) and 400 μg·ml⁻¹ of Au-MNPs. The reaction wasinitiated with 0.2 mM of H₂O₂ and incubated for 24 hours. The BNCsincrease the release of aromatics from biomass.

FIGS. 8A-8C. Spectra showing use of manganese peroxidase and versatileperoxidase in the presence of glucose oxidase for lignindepolymerization of biomass feedstock (mixed grasses). The glucoseoxidase produces hydrogen peroxide in the presence of glucose. Correctedspectra of biomass hydrolysates at 12 hours for differentperoxidase-to-oxidase molar ratio. BNCs were formed with manganeseperoxidase and versatile peroxidase at 25 nM each-15 μg·ml⁻¹ Au-MNPs).FIG. 8A: Glucose oxidase was at 6.125 nM (4A: ratio of 4); FIG. 8B: 12.5nM (4B: ratio of 2), and FIG. 8C: 25 nM (4C: ratio of 1). The BNCs canaccommodate an in situ hydrogen production system to increase therelease of aromatics from biomass and use glucose as oxidant.

FIG. 9. Schematic showing the assembly of BNCs of horseradish peroxidasetemplates on magnetite microparticles to form hierarchical macroporouscatalysts for applications including phenol remediation and chemicalconversion of aromatics.

FIG. 10. Graph showing extent of phenol removal using BNC and BMC after12 hours at room temperature at equimolar concentration of phenol andH₂O₂ (1 mM). The error bars are the standard deviation of triplicates.The templating of the BNCs onto microparticles to form mesoporouscatalysts is not detrimental to the activity of the BNCs.

FIGS. 11A, 11B. Bar charts showing reuse performance of catalysts aftereach cycle for five cycles for phenol removal: [phenol]=[H₂O₂]=1 mM;reaction time 2 hours at room temperature; [HRP]=30 nM, [MNP]=60 ug/ml.FIG. 11A is for [MMP]/[MNP]/[HRP]=20:2:1, and FIG. 11B is for[MMP]/[MNP]/[HRP]=160:2:1. The error bars are the standard deviation oftriplicates. The hierarchical structures allow the reuse of the enzymecatalysts for several cycles.

FIG. 12A, 12B. Scanning electronic microscope (SEM) images of thehorseradish peroxidase BNCs templated on magnetite microparticles andtwo different ratios of BNCs. The assemblies of BNCs onto the surface ofmicroparticles is a self-assembling process driven by magneticinteractions.

FIG. 13. Photograph showing a V-shape conformation of the magneticreactors; the electromagnets are paired and cycled on a 1-2 basis. Theprototype system has been designed with electromagnets (tubular 9.6×16.7mm, Series E-66-38, Magnetic Sensor Systems, CA), a simplemicrocontroller and interface circuits between the microcontroller andelectromagnets. The electromagnets are mounted on a custom stand, whichallows them to be positioned in various configurations at the side of asmall bioreactor consisting of disposable UV transparent plasticmicro-cuvettes with a 0.5 cm cross-section.

FIG. 14. Photograph showing an I-shape conformation of the magneticreactors; the electromagnets are paired and cycled on a 1-1 basis. Thepink color is characteristic of the synthesis of quinoneimine fromphenol and aminopyrene using high-efficiency horseradishperoxidase-based magnetic catalysts. This reaction illustrates theremoval of phenols and the synthesis chemistry of pigments, dyes, aromasand fine chemicals.

FIG. 15. Schematic showing the use of BNCs for phenol removal byoxidative polymerization. The phenol molecules are oxidized tophenoxy-radicals that polymerize with each other. The reaction ischaracteristic of the remediation of aromatics and the polymerization byoxidative coupling.

FIG. 16. Schematic showing the integration of magnetic reactors forwater treatment.

FIG. 17. Schematic showing the core of the magnetic reactors for thedecoupling of free radical generation and polymerization.

DETAILED DESCRIPTION OF THE DISCLOSURE

In one aspect, the invention is directed to an enzyme-containingcomposition that includes mesoporous aggregates of magneticnanoparticles adsorbed to one or more enzymes, wherein the one or moreenzymes may or may not include a free-radical producing (FRP) enzyme.The assembly of magnetic nanoparticles adsorbed to enzyme is herein alsoreferred to as a “bionanocatalyst” or “BNC”. As used herein, the term“adsorbed” is meant to be synonymous with the terms “bound”, “associatedwith”, or “aggregated with”, as long as the mode of attachment preventsor substantially minimizes release of the enzyme from the magneticnanoparticles under conditions in which they are used or stored forlater use. The BNCs or noble-metal coated versions thereof may also beadsorbed onto (i.e., be made to reside on) the surface of a macroporousscaffold, which may be an assembly of magnetic microparticles and/or anyof the continuous macroporous scaffolds described below.

The BNC contains mesopores that are interstitial spaces between themagnetic nanoparticles. The enzyme may be located anywhere on themagnetic nanoparticle, e.g., on the surface and/or embedded within atleast a portion of mesopores of the BNC. As used herein, the term“magnetic” encompasses all types of useful magnetic characteristics,including permanent magnetic, superparamagnetic, paramagnetic,ferromagnetic, and ferrimagnetic behaviors.

The magnetic nanoparticle or BNC has a size in the nanoscale, i.e.,generally no more than 500 nm. As used herein, the term “size” can referto a diameter of the magnetic nanoparticle when the magneticnanoparticle is approximately or substantially spherical. In a casewhere the magnetic nanoparticle is not approximately or substantiallyspherical (e.g., substantially ovoid or irregular), the term “size” canrefer to either the longest the dimension or an average of the threedimensions of the magnetic nanoparticle. The term “size” may also referto an average of sizes over a population of magnetic nanoparticles(i.e., “average size”). In different embodiments, the magneticnanoparticle has a size of precisely, about, up to, or less than, forexample, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 50 nm, 40 nm, 30 nm, 25nm, 20 nm, 15 nm, 10 nm, 5 nm, 4 nm, 3 nm, 2 nm, or 1 nm, or a sizewithin a range bounded by any two of the foregoing exemplary sizes.

In the BNC, the individual magnetic nanoparticles can be considered tobe primary nanoparticles (i.e., primary crystallites) having any of thesizes provided above. The aggregates of nanoparticles in a BNC arelarger in size than the nanoparticles and generally have a size (i.e.,secondary size) of at least 5 nm. In different embodiments, theaggregates have a size of precisely, about, at least, above, up to, orless than, for example, 5 nm, 8 nm, 10 nm, 12 nm, 15 nm, 20 nm, 25 nm,30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm,150 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, or 800 nm, or asize within a range bounded by any two of the foregoing exemplary sizes.

Typically, the primary and/or aggregated magnetic nanoparticles or BNCsthereof have a distribution of sizes, i.e., they are generally dispersedin size, either narrowly or broadly dispersed. In different embodiments,any range of primary or aggregate sizes can constitute a major or minorproportion of the total range of primary or aggregate sizes. Forexample, in some embodiments, a particular range of primary particlesizes (for example, at least 1, 2, 3, 5, or 10 nm and up to 15, 20, 25,30, 35, 40, 45, or 50 nm) or a particular range of aggregate particlesizes (for example, at least 5, 10, 15, or 20 nm and up to 50, 100, 150,200, 250, or 300 nm) constitutes at least or above 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% of the total range of primary particlesizes. In other embodiments, a particular range of primary particlesizes (for example, less than 1, 2, 3, 5, or 10 nm, or above 15, 20, 25,30, 35, 40, 45, or 50 nm) or a particular range of aggregate particlesizes (for example, less than 20, 10, or 5 nm, or above 25, 50, 100,150, 200, 250, or 300 nm) constitutes no more than or less than 50%,40%, 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% of the total range ofprimary particle sizes.

The aggregates of magnetic nanoparticles (i.e., “aggregates”) or BNCsthereof can have any degree of porosity, including a substantial lack ofporosity depending upon the quantity of individual primary crystallitesthey are made of. In particular embodiments, the aggregates aremesoporous by containing interstitial mesopores (i.e., mesopores locatedbetween primary magnetic nanoparticles, formed by packing arrangements).The mesopores are generally at least 2 nm and up to 50 nm in size. Indifferent embodiments, the mesopores can have a pore size of preciselyor about, for example, 2, 3, 4, 5, 10, 12, 15, 20, 25, 30, 35, 40, 45,or 50 nm, or a pore size within a range bounded by any two of theforegoing exemplary pore sizes. Similar to the case of particle sizes,the mesopores typically have a distribution of sizes, i.e., they aregenerally dispersed in size, either narrowly or broadly dispersed. Indifferent embodiments, any range of mesopore sizes can constitute amajor or minor proportion of the total range of mesopore sizes or of thetotal pore volume. For example, in some embodiments, a particular rangeof mesopore sizes (for example, at least 2, 3, or 5, and up to 8, 10,15, 20, 25, or 30 nm) constitutes at least or above 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% of the total range of mesopore sizes or ofthe total pore volume. In other embodiments, a particular range ofmesopore sizes (for example, less than 2, 3, 4, or 5 nm, or above 10,15, 20, 25, 30, 35, 40, 45, or 50 nm) constitutes no more than or lessthan 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% of the totalrange of mesopore sizes or of the total pore volume.

The magnetic nanoparticles can have any of the compositions known in theart. In some embodiments, the magnetic nanoparticles are or include azerovalent metallic portion that is magnetic. Some examples of suchzerovalent metals include cobalt, nickel, and iron, and their mixturesand alloys. In other embodiments, the magnetic nanoparticles are orinclude an oxide of a magnetic metal, such as an oxide of cobalt,nickel, or iron, or a mixture thereof. In some embodiments, the magneticnanoparticles possess distinct core and surface portions. For example,the magnetic nanoparticles may have a core portion composed of elementaliron, cobalt, or nickel and a surface portion composed of a passivatinglayer, such as a metal oxide or a noble metal coating, such as a layerof gold, platinum, palladium, or silver. In other embodiments, metaloxide magnetic nanoparticles or aggregates thereof are coated with alayer of a noble metal coating. The noble metal coating may, forexample, reduce the number of charges on the magnetic nanoparticlesurface, which may beneficially increase dispersibility in solution andbetter control the size of the BNCs. The noble metal coating protectsthe magnetic nanoparticles against oxidation, solubilization by leachingor by chelation when chelating organic acids, such as citrate, malonate,or tartrate, are used in the biochemical reactions or processes. Thepassivating layer can have any suitable thickness, and particularly, atleast, up to, or less than, for example, 0.1 nm, 0.2 nm, 0.3 nm, 0.4 nm,0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6nm, 7 nm, 8 nm, 9 nm, or 10 nm, or a thickness in a range bounded by anytwo of these values.

In particular embodiments, the magnetic nanoparticles have an iron oxidecomposition. The iron oxide composition can be any of the magnetic orsuperparamagnetic iron oxide compositions known in the art, e.g.,magnetite (Fe₃O₄), hematite (α-Fe₂O₃), maghemite (γ-Fe₂O₃), or a spinelferrite according to the formula AB₂O₄, wherein A is a divalent metal(e.g., Zn²⁺, Ni²⁺, Mn²⁺, Co²⁺, Ba²⁺, Sr²⁺, or combination thereof) and Bis a trivalent metal (e.g., Fe³⁺, Cr³⁺, or combination thereof).

In particular embodiments, the above mesoporous aggregates of magneticnanoparticles (BNCs) are incorporated into a continuous macroporousscaffold to form a hierarchical catalyst assembly with first and secondlevels of assembly. The first level of assembly is found in the BNCs.The second level of assembly is found in the incorporation of the BNCsinto the continuous macroporous scaffold. The overall hierarchicalcatalyst assembly is magnetic by at least the presence of the BNCs.

The term “continuous”, as used herein for the macroporous scaffold,indicates a material that is not a particulate assembly, i.e., is notconstructed of particles or discrete objects assembled with each otherto form a macroscopic structure. In contrast to a particulate assembly,the continuous structure is substantially seamless and uniform aroundmacropores that periodically interrupt the seamless and uniformstructure. The macropores in the continuous scaffold are, thus, notinterstitial spaces between agglomerated particles. Nevertheless, thecontinuous scaffold can be constructed of an assembly or aggregation ofsmaller primary continuous scaffolds, as long as the assembly oraggregation of primary continuous scaffolds does not include macropores(e.g., from above 50 nm and up to 100 microns) formed by interstitialspaces between primary continuous scaffolds. Particularly in the case ofinorganic materials, such as ceramics or elemental materials, thecontinuous scaffold may or may not also include crystalline domains orphase boundaries.

The macroporous scaffold contains macropores (i.e., pores of amacroscale size) having a size greater than 50 nm. In differentembodiments, the macropores have a size of precisely, about, at least,above, up to, or less than, for example, 60 nm, 70 nm, 80 nm, 90 nm, 100nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900nm, 1 micron (1 μm), 1.2 μn, 1.5 μn, 2 μn, 3 μn, 4 μn, 5 μn, 10 μn, 20μn, 30 μn, 40 μn, 50 μn, 60 μn, 70 μn, 80 μn, 90 μn, or 100 μn, or asize within a range bounded by any two of the foregoing exemplary sizes.

The macroporous scaffold can have any suitable size as long as it canaccommodate macropores. In typical embodiments, the macroporous scaffoldpossesses at least one size dimension in the macroscale. The at leastone macroscale dimension is above 50 nm, and can be any of the valuesprovided above for the macropores, and in particular, a dimension ofprecisely, about, at least, above, up to, or less than, for example, 1μn, 2 μn, 3 μn, 4 μn, 5 μn, 10 μn, 20 μn, 30 μn, 40 μn, 50 μn, 60 μn, 70μn, 80 μn, 90 μn, 100 μn, 200 μn, 300 μn, 400 μn, 500 μn, 600 μn, 700μn, 800 μn, 900 μn, 1 mm, 2 mm, 5 mm, or 1 cm, or a size within a rangebounded by any two of the foregoing exemplary sizes. Where only one ortwo of the size dimensions are in the macroscale, the remaining one ortwo dimensions can be in the nanoscale, such as any of the valuesprovided above for the magnetic nanoparticles (e.g., independently,precisely, about, at least, above, up to, or less than, for example, 1,2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nm, or a valuewithin a range bounded by any two of the foregoing values). In someembodiments, at least two or all of the size dimensions of themacroporous scaffold is in the macroscale.

In a first set of embodiments, the continuous macroporous scaffold inwhich the BNCs are incorporated is magnetic, i.e., even in the absenceof the BNCs. The continuous macroporous scaffold can be magnetic by, forexample, being composed of a magnetic polymer composition. An example ofa magnetic polymer is PANiCNQ, which is a combination oftetracyanoquinodimethane (TCNQ) and the emeraldine-based form ofpolyaniline (PANi), as well known in the art. Alternatively, or inaddition, the continuous macroporous scaffold can be magnetic by havingembedded therein magnetic particles not belonging to the BNCs. Themagnetic particles not belonging to the BNCs may be, for example,magnetic nano- or micro-particles not associated with an FRP enzyme orany enzyme. The magnetic microparticles may have a size or sizedistribution as provided above for the macropores, although independentof the macropore sizes. In particular embodiments, the magneticmicroparticles have a size of about, precisely, or at least 20, 30, 40,50, 60, 70, 80, 90, 100, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000 nm, or a size within a range bounded by any two of the foregoingexemplary sizes. In some embodiments, the continuous macroporousscaffold has embedded therein magnetic microparticles that are adsorbedto at least a portion of the BNCs, or wherein the magneticmicroparticles are not associated with or adsorbed to the BNCs.

In a second set of embodiments, the continuous scaffold in which theBNCs are incorporated is non-magnetic. Nevertheless, the overallhierarchical catalyst assembly containing the non-magnetic scaffoldremains magnetic by at least the presence of the BNCs incorporatedtherein.

In one embodiment, the continuous macroporous scaffold (or precursorthereof) has a polymeric composition. The polymeric composition can beany of the solid organic, inorganic, or hybrid organic-inorganic polymercompositions known in the art, and may be synthetic or a biopolymer thatacts as a binder. Preferably, the polymeric macroporous scaffold doesnot dissolve or degrade in water or other medium in which thehierarchical catalyst is intended to be used. Some examples of syntheticorganic polymers include the vinyl addition polymers (e.g.,polyethylene, polypropylene, polystyrene, polyacrylic acid orpolyacrylate salt, polymethacrylic acid or polymethacrylate salt,poly(methylmethacrylate), polyvinyl acetate, polyvinyl alcohol, and thelike), fluoropolymers (e.g., polyvinylfluoride, polyvinylidenefluoride,polytetrafluoroethylene, and the like), the epoxides (e.g., phenolicresins, resorcinol—formaldehyde resins), the polyamides, thepolyurethanes, the polyesters, the polyimides, the polybenzimidazoles,and copolymers thereof. Some examples of biopolymers include thepolysaccharides (e.g., cellulose, hemicellulose, xylan, chitosan,inulin, dextran, agarose, and alginic acid), polylactic acid, andpolyglycolic acid. In the particular case of cellulose, the cellulosemay be microbial- or algae-derived cellulose. Some examples of inorganicor hybrid organic-inorganic polymers include the polysiloxanes (e.g., asprepared by sol gel synthesis, such as polydimethylsiloxane) andpolyphosphazenes. In some embodiments, any one or more classes orspecific types of polymer compositions provided above are excluded asmacroporous scaffolds.

In another embodiment, the continuous macroporous scaffold (or precursorthereof) has a non-polymeric composition. The non-polymeric compositioncan have, for example, a ceramic or elemental composition. The ceramiccomposition may be crystalline, polycrystalline, or amorphous, and mayhave any of the compositions known in the art, including oxidecompositions (e.g., alumina, beryllia, ceria, yttria, or zirconia) andnon-oxide compositions (e.g., carbide, silicide, nitride, boride, orsulfide compositions). The elemental composition may also becrystalline, polycrystalline, or amorphous, and may have any suitableelemental composition, such as carbon, aluminum, or silicon.

In other embodiments, the BNCs reside in a non-continuous macroporoussupport containing (or constructed of) an assembly (i.e., aggregation)of magnetic microparticles (MMPs) that includes macropores asinterstitial spaces between the magnetic microparticles. The magneticmicroparticles are typically ferromagnetic. The BNCs are embedded in atleast a portion of the macropores of the aggregation of magneticmicroparticles, and may also reside on the surface of the magneticmicroparticles. The BNCs can associate with the surface of the magneticmicroparticles by magnetic interaction. The magnetic microparticles mayor may not be coated with a metal oxide or noble metal coating layer. Insome embodiments, the BNC-MMP assembly is incorporated (i.e., embedded)into a continuous macroporous scaffold, as described above, to provide ahierarchical catalyst assembly.

The individual magnetic nanoparticles or aggregates thereof or BNCsthereof possess any suitable degree of magnetism. For example, themagnetic nanoparticles, BNCs, or BNC-scaffold assemblies can possess asaturated magnetization (M_(s)) of at least or up to 5, 10, 15, 20, 25,30, 40, 45, 50, 60, 70, 80, 90, or 100 emu/g. The magneticnanoparticles, BNCs, or BNC-scaffold assemblies preferably possess aremanent magnetization (M_(r)) of no more than (i.e., up to) or lessthan 5 emu/g, and more preferably, up to or less than 4 emu/g, 3 emu/g,2 emu/g, 1 emu/g, 0.5 emu/g, or 0.1 emu/g. The surface magnetic field ofthe magnetic nanoparticles, BNCs, or BNC-scaffold assemblies can beabout or at least, for example, 0.5, 1, 5, 10, 50, 100, 200, 300, 400,500, 600, 700, 800, 900, or 1000 Gauss (G), or a magnetic field within arange bounded by any two of the foregoing values. If microparticles areincluded, the microparticles may also possess any of the above magneticstrengths.

The magnetic nanoparticles or aggregates thereof can be made to adsorb asuitable amount of enzyme, up to or below a saturation level, dependingon the application, to produce the resulting BNC. In differentembodiments, the magnetic nanoparticles or aggregates thereof may adsorbabout, at least, up to, or less than, for example, 1, 5, 10, 15, 20, 25,or 30 pmol/m² of enzyme. Alternatively, the magnetic nanoparticles oraggregates thereof may adsorb an amount of enzyme that is about, atleast, up to, or less than, for example, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 100% of a saturation level.

The magnetic nanoparticles or aggregates thereof or BNCs thereof possessany suitable pore volume. For example, the magnetic nanoparticles oraggregates thereof can possess a pore volume of about, at least, up to,or less than, for example, 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35,0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1cm³/g, or a pore volume within a range bounded by any two of theforegoing values.

The magnetic nanoparticles or aggregates thereof or BNCs thereof possessany suitable specific surface area. For example, the magneticnanoparticles or aggregates thereof can have a specific surface area ofabout, at least, up to, or less than, for example, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 m²/g.

For purposes of the invention, the enzyme preferably functions byconverting a diffusible substrate into a diffusible product. The enzymecan be from any source, e.g., fungal, microbial, animal, or plant.

In particular embodiments, the enzyme has the property of producing freeradicals, i.e., is a “FRP enzyme”. In particular embodiments, the FRPenzyme is an oxidoreductase belonging to the EC 1 family of enzymes. TheEC 1 oxidoreductase can be, for example, an EC 1.1 oxidoreductase actingon the CH—OH groups of donors, an EC 1.2 oxidoreductase acting on thealdehyde or oxo group of donors, an EC 1.3 oxidoreductase acting on theCH—CH group of donors, an EC 1.4 oxidoreductase acting on the CH—NH₂group of donors, an EC 1.5 oxidoreductase acting on the CH—NH group ofdonors, an EC 1.6 oxidoreductase acting on NADH or NADPH, an EC 1.7oxidoreductase acting on various nitrogenous compounds as donors, an EC1.8 oxidoreductase acting on a sulfur group as donor, an EC 1.9oxidoreductase acting on a heme group of donors, an EC 1.10oxidoreductase acting on diphenols and related substances as donors, anEC 1.11 oxidoreductase acting on peroxide as an acceptor, an EC 1.12oxidoreductase acting on hydrogen as a donor, an EC 1.13 oxidoreductaseacting on single donors with incorporation of molecular oxygen(oxygenases), an EC 1.14 oxidoreductase acting on paired donors withincorporation or reduction of molecular oxygen, an EC 1.15oxidoreductase acting on superoxide as an acceptor, an EC 1.16oxidoreductase that oxidize metal ions, an EC 1.17 oxidoreductase actingon CH or CH₂ groups, an EC 1.18 oxidoreductase acting on iron-sulfurproteins as a donor, an EC 1.19 oxidoreductase acting on reducedflavodoxin as a donor, an EC 1.20 oxidoreductase acting on phosphorus orarsenic as a donor, an EC 1.21 oxidoreductase acting on X—H and Y—H toform an X—Y bond, an EC 1.97 oxidoreductase, an EC 1.98 oxidoreductasethat uses hydrogen as a reductant, and an EC 1.99 oxidoreductase thatuses oxygen as an oxidant. The oxidoreductase may also be moreparticularly identified as belonging to a sub-genus of any of the EC 1.1groupings provided above.

In a first particular set of embodiments, the FRP enzyme is selectedfrom the EC 1.1 genus of oxidoreductase enzymes. The EC 1.1 enzyme canfurther be identified as belonging to any of the following sub-genuses:EC 1.1.1 with NAD or NADP as acceptor, EC 1.1.2 with a cytochrome asacceptor, EC 1.1.3 with oxygen as acceptor, EC 1.1.4 with disulfide asacceptor, EC 1.1.5 with quinone or similar compound as acceptor, and EC1.1.99 with other acceptors. In more particular embodiments, the FRPenzyme is identified as belonging to a sub-genus of any of the EC 1.1sub-genuses provided above. For example, the FRP enzyme can beidentified as belonging to any of the sub-genuses of EC 1.1.3, such asEC 1.1.3.3 (malate oxidase), EC 1.1.3.4 (glucose oxidase), EC 1.1.3.5(hexose oxidase), EC 1.1.3.6 (cholesterol oxidase), EC 1.1.3.7(aryl-alcohol oxidase), EC 1.1.3.8 (L-gulonolactone oxidase), EC 1.1.3.9(galactose oxidase), EC 1.1.3.10 (pyranose oxidase), EC 1.1.3.11(L-sorbose oxidase), EC 1.1.3.12 (pyridoxine 4-oxidase), EC 1.1.3.13(alcohol oxidase), EC 1.1.3.14 (catechol oxidase), EC 1.1.3.15(2-hydroxy acid oxidase), EC 1.1.3.16 (ecdysone oxidase), EC 1.1.3.17(choline oxidase), EC 1.1.3.18 (secondary-alcohol oxidase), EC 1.1.3.19(4-hydroxymandelate oxidase), EC 1.1.3.20 (long-chain alcohol oxidase),EC 1.1.3.21 (glycerol-3-phosphate oxidase), EC 1.1.3.22, EC 1.1.3.23(thiamine oxidase), EC 1.1.3.24 (L-galactonolactone oxidase), EC1.1.3.25, EC 1.1.3.26, EC 1.1.3.27 (hydroxyphytanate oxidase), EC1.1.3.28 (nucleoside oxidase), EC 1.1.3.29 (N-acylhexosamine oxidase),EC 1.1.3.30 (polyvinyl alcohol oxidase), EC 1.1.3.31, EC 1.1.3.32, EC1.1.3.33, EC 1.1.3.34, EC 1.1.3.35, EC 1.1.3.36, EC 1.1.3.37D-arabinono-1,4-lactone oxidase), EC 1.1.3.38 (vanillyl alcoholoxidase), EC 1.1.3.39 (nucleoside oxidase, H₂O₂ forming), EC 1.1.3.40(D-mannitol oxidase), and EC 1.1.3.41 (xylitol oxidase).

In a second particular set of embodiments, the FRP enzyme is selectedfrom the EC 1.10 genus of oxidoreductase enzymes. The EC 1.10 enzyme canfurther be identified as belonging to any of the following sub-genuses:EC 1.10.1 with NAD or NADP as acceptor EC 1.10.2 with cytochrome asacceptor, EC 1.10.3 with oxygen as acceptor, and EC 1.10.99 with otheracceptors. The EC 1.10.1 enzyme can be more specifically, for example,EC 1.10.1.1, i.e., trans-acenaphthene-1,2-diol dehydrogenase. The EC1.10.2 enzyme can be more specifically, for example, EC 1.10.2.1(cytochrome-b5 reductase) or EC 1.10.2.2 (cytochrome-c reductase). TheEC 1.10.3 enzyme can be more specifically, for example, EC 1.10.3.1(catechol oxidase), EC 1.10.3.2 (laccase), EC 1.10.3.3 (L-ascorbateoxidase), EC 1.10.3.4 (o-aminophenol oxidase), EC 1.10.3.5(3-hydroxyanthranilate oxidase), EC 1.10.3.6 (rifamycin-B oxidase), EC1.10.3.7, or EC 1.10.3.8. The EC 1.10.99 enzyme can be morespecifically, for example, EC 1.10.99.1 (plastoquinol-plastocyaninreductase), EC 1.10.99.2 (ribosyldihydronicotinamide dehydrogenase,quinone), or EC 1.10.99.3 (violaxanthin de-epoxidase).

In a third particular set of embodiments, the FRP enzyme is selectedfrom the EC 1.11 genus of oxidoreductase enzymes. The EC 1.11 enzyme canfurther be identified as belonging to the sub-genus EC 1.11.1(peroxidases). The EC 1.11.1 enzyme can be more specifically, forexample, EC 1.11.1.1 (NADH peroxidase), EC 1.11.1.2 (NADPH peroxidase),EC 1.11.1.3 (fatty acid peroxidase), EC 1.11.1.4, EC 1.11.1.5(cytochrome-c peroxidase), EC 1.11.1.6 (catalase), EC 1.11.1.7(peroxidase), EC 1.11.1.8 (iodide peroxidase), EC 1.11.1.9 (glutathioneperoxidase), EC 1.11.1.10 (chloride peroxidase), EC 1.11.1.11(L-ascorbate peroxidase), EC 1.11.1.12 (phospholipid-hydroperoxideglutathione peroxidase), EC 1.11.1.13 (manganese peroxidase), EC1.11.1.14 (diarylpropane peroxidase), or EC 1.11.1.15 (peroxiredoxin).

In particular embodiments, the FRP enzyme is a peroxidase. Theperoxidase may also be further specified by function, e.g., a ligninperoxidase, manganese peroxidase, or versatile peroxidase. Theperoxidase may also be specified as a fungal, microbial, animal, orplant peroxidase. The peroxidase may also be specified as a class I,class II, or class III peroxidase. The peroxidase may also be specifiedas a myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase(LPO), thyroid peroxidase (TPO), prostaglandin H synthase (PGHS),glutathione peroxidase, haloperoxidase, catalase, cytochrome cperoxidase, horseradish peroxidase, peanut peroxidase, soybeanperoxidase, turnip peroxidase, tobacco peroxidase, tomato peroxidase,barley peroxidase, or peroxidasin. In particular embodiments, theperoxidase is horseradish peroxidase.

In some embodiments, a single enzyme is used, which may or may not be aFRP enzyme. In other embodiments, a combination of enzymes is used,which may or may not include a FRP enzyme. The combination of enzymescan be, for example, any two or three oxidoreductase enzymes selectedfrom any of the above classes or sub-classes therein. In someembodiments, a combination of FRP enzymes (e.g., EC 1 enzymes) is used.In particular embodiments, a combination of EC 1.1 enzymes is used. Inother particular embodiments, a combination of EC 1.10 enzymes is used.In other particular embodiments, a combination of EC 1.11 enzymes isused. In other embodiments, a combination of any of the particular FRPenzymes described above and a peroxidase is used (e.g., a combination ofa EC 1.1 or EC 1.1.3 enzyme and a peroxidase). When a combination of FRPenzymes is used, the two or more enzymes may be arranged in a core-shelltype of arrangement, i.e., a first FRP enzyme is either in a coreportion or surface portion of the magnetic nanoparticle or aggregatethereof, and a second (different) FRP enzyme covers the region where thefirst FRP enzyme is located. The second FRP enzyme may be an aggregateof the magnetic nanoparticle or on the surface thereof, overlaying thefirst enzyme.

In the case of multiple enzyme systems, manipulating the distribution ofthe different enzymes within the mesoporous aggregates offers theadvantage of decoupling the different reactions and permitting diffusionof the substrates and products of the reactions from one layer toanother layer or to the core of the BNCs. Therefore, when performing theenzymatic reactions in the confined pore structures of the BNCs,core/shell distributions offer the possibility of better controlling thekinetics of the different entrapped FRP enzymes. Combining enzymes thatperform similar reactions (such as two, or more, peroxidases or aperoxidase and a laccase for example) but having different reactionrequirements (substrates, substrate concentration, etc.) canbeneficially increase the versatility of the BNCs to perform in broadand variable process conditions at a high level of efficiency. Combiningenzymes with coupled reactions can ensure the production of thesubstrate in the vicinity of the enzyme and bypass the need forhazardous and labile chemical substrates, such as hydrogen peroxide. Forexample, a glucose oxidase enzyme can generate hydrogen peroxide fromglucose, which is an inexpensive and non-hazardous compound.

In another aspect, the invention is directed to methods for producingthe mesoporous aggregates of magnetic nanoparticles with enzyme embeddedtherein (BNCs). In particular embodiments, the BNCs are prepared bycombining soluble enzymes with a monodispersed solution of magneticnanoparticles, which may or may not be coated. The monodispersed stateof the nanoparticle prior to mixing can be achieved by sonication of thenanoparticles. Enzymes and monodispersed nanoparticles are incubatedunder permanent agitation until all enzymes are adsorbed and clustersform. The pH of the solution for BNC synthesis needs to be such that theoverall electrostatic charge of surface groups the enzymes is oppositeto the overall electrostatic charge of the surface of the nanoparticles.For optimal formation of BNCs, the pH of the solution should be adjustedto prevent self-aggregation of the nanoparticles, and the presence ofcounterions is generally undesirable. In one embodiment, for optimalformation of BNCs, the surface potential of the enzymes (pKa) and thenanoparticles is no more than three units to limit over-aggregation andclumping. In another embodiment, for optimal formation of BNCs, theconcentration of enzymes is no more than about 80% of the total bindingcapacity of the nanoparticle surface in order to preventover-aggregation. The size of the BNC clusters is related to the ratioof nanoparticles vs. enzyme in solution. Generally, the more enzyme, thelarger the clusters become. For optimal activities and to limitdiffusion hindrance of the substrates and products, the clusters shouldgenerally be larger than about 200 nm.

The magnetic nanoparticles or aggregates thereof or BNCs thereof mayalso be coated with a noble metal, such as gold, platinum, or palladium.Alternatively, or in addition, the magnetic nanoparticles or aggregatesthereof or BNCs thereof may be coated with a metal oxide layer (e.g.,silica or titania) or polymeric protective coating to protect againstoxidation of the magnetic nanoparticles. Any suitable method for coatingthe magnetic nanoparticles may be used. For example, in particularembodiments, magnetic nanoparticles are dispersed in a solutioncontaining a noble metal salt, and the noble metal salt subjected toreducing conditions. The foregoing method can be facilitated by bindingdifunctional molecules onto the surface of the magnetic nanoparticlesbefore the noble metal salt is reduced. The bifunctional molecules usedfor this purpose should contain a portion useful for binding to themagnetic nanoparticles (as described above) as well as a noble metalbinding portion (e.g., an amine, thiol, phosphine, or chelating moiety)for binding noble metal ions. Optionally, once metal ions are bound tothe nanoparticle surface, the magnetic nanoparticles can be washed ofexcess noble metal salt (e.g., by magnetic capture, filtration, ordecanting). Since noble metal ions are attached to the surface, theforegoing methodology provides a more selective method for producing anoble metal coating (i.e., without concomitant production of noble metalnanoparticles) as well as a more uniform coating. In some embodiments,the noble metal coating is applied before enzyme is included with themagnetic nanoparticles, in which case enzyme is later bonded to thenoble metal coating. The enzyme can be bonded to the noble metal coatingby, for example, functionalizing the noble metal coating withdifunctional molecules that bind to the noble metal coating and possessanother reactive group for binding to the enzyme.

In another aspect, the invention is directed to a method for producing ahierarchical catalyst assembly containing the BNCs incorporated into amacroporous scaffold, as described above. In typical embodiments, theBNCs or noble-metal coated versions thereof, are made to adsorb onto thesurface of a macroporous scaffold by contacting the BNCs or noble-metalcoated versions thereof with the macroporous scaffold in anaqueous-based solution such that the nanoparticles adsorb onto thesurface of the macroporous scaffold. In the method, BNCs are generallycontacted with the macroporous scaffold in solution (i.e., liquidsolution) to substantially embed the BNCs into macropores of thescaffold. The solution can include water and/or any suitable solventthat permits efficient and intimate contact between the BNCs andscaffold. Typically, the BNCs will adsorb onto or into the scaffold byself-assembly mechanisms, i.e., by magnetic interaction, physisorption,and/or chemisorption. After the BNCs have been embedded into thescaffold, the catalyst assembly may be used without further processing,or the catalyst assembly may be rinsed in water or a suitable solvent,or stored before use, e.g., in a solution suitable for preserving theenzyme. In some embodiments, the BNCs are adhered onto the surface ofmagnetic microparticles, and the BNC-microparticle assembly embeddedinto a continuous macroporous scaffold.

The templating of the BNCs onto macroporous scaffolds can be performedin the same buffer as used for BNC formation. Small quantities ofmacroporous scaffolds are typically added sequentially to the BNCs untilthe supernatant is clear, which indicates that all the BNCs have beentrapped on the surface of the scaffolds. The color of the supernatant isgenerally monitored by the absorbance of the nanoparticle clusters aftercapture of the BNC-loaded scaffolds with small electromagnets. Thequantity of scaffold needed to capture BNCs depends on the massmagnetization and the surface area of the scaffold. Alternatively, thebinding capacity of the scaffold can be determined and the appropriateamount of BNCs added for complete capture. To increase the massmagnetization of the material in solution, BNC-functionalized scaffoldscan be diluted with non-functionalized scaffolds without changing theconcentration of the enzyme needed for the process.

A continuous macroporous scaffold can be prepared by any suitablemethod. Any process known in the art for incorporating macropores into amaterial are considered herein. In particular embodiments, thecontinuous macroporous scaffold is produced by a templating (temptation)process that includes: (i) producing a composite containing a scaffoldprecursor material or composition having a sacrificial templating agentembedded therein, and (ii) selective removal of the sacrificialtemplating agent to produce macropores in the scaffold precursormaterial. In a first set of embodiments, the templating process employsa solvent as the templating agent, wherein the solvent is embedded inthe scaffold precursor material. In the solvent temptation process, thecomposite containing the scaffold precursor material with embeddedsolvent is cooled until the embedded solvent freezes to form solventcrystals, and then the frozen solvent is removed by either evaporationor sublimation to produce macropores in the scaffold precursor material.When the solvent is water, the solvent temptation process can bereferred to as an ice temptation process. In a second set ofembodiments, the templating process employs a solid sacrificialtemplating agent that is embedded in the scaffold precursor material.The sacrificial templating agent can be, for example, a polymeric ormetal oxide substance that can be selectively removed afterincorporation into the scaffold precursor. The incorporation of suchsacrificial templating agents are well known in the art. By methods wellknown in the art, the sacrificial templating agent can be selectivelyremoved by, for example, acid or base leaching, solvent dissolution, orpyrolytic decomposition. In other embodiments, the sacrificialtemplating agent is a burn-out material, which is a material that eithervolatilizes or decomposes upon application of sufficient heat to producethe macropores.

Further details of macropore-forming methods conventionally used in theart can be found in, for example, L. Yang, et al., “Robust MacroporousMaterials of Chiral Polyaniline Composites”, Chem. Mater., 18(2), pp.297-3000 (2006); M. Abdullah, et al., “Preparation of Oxide Particleswith Ordered Macropores by Colloidal Templating and Spray Pyrolysis”,Acta Materialia, 52, pp. 5151-5156 (2004); T. Niu et al., “Preparationof Meso-Macroporous A-Alumina Using Carbon Nanotube as the Template forthe Mesopore and Their Application to the Preferential Oxidation of COin H₂-Rich Gases”, Journal of Porous Materials, vol. 20, issue 4, pp.789-798 (2013); and M. Davis, et al., “Formation of Three-DimensionalOrdered Hierarchically Porous Metal Oxides via a Hybridized EpoxideAssisted/Colloidal Crystal Templating Approach”, ACS Appl. Mater.Interfaces, 5(16), pp. 7786-7792 (2013), all of which are hereinincorporated by reference in their entirety.

FIGS. 1A and 1B depict an exemplary process for producing first andsecond levels of assembly, respectively, in a hierarchical catalystassembly. FIG. 1A shows an exemplary process for forming a first levelof assembly of a hierarchical catalyst assembly containing BNCs composedof mesoporous aggregates of magnetic nanoparticles and horseradishperoxidase. FIG. 1B shows an exemplary process for forming a secondlevel of assembly by incorporating the BNCs of FIG. 1A into amacroporous scaffold composed of an ice-templated continuous material,such as a polymeric material, such as a biopolymer, which may be apolysaccharide, such as cellulose. In some embodiments, the scaffoldmaterial may be sonicated before use in order to untangle or disperseindividual sheets, fibers, or ribbons of the material. Theice-templating approach described above can be applied to otherpolymers, such as, for example, chitosan, agars, and polymeric resins.

In another aspect, the invention is directed to a process fordepolymerizing lignin, i.e., a lignin depolymerization process, in whichany of the BNCs or BNC-scaffold structures described above is used fordepolymerizing or facilitating the depolymerization of lignin. Thelignin being depolymerized can be any lignin-containing material. Theprecursor lignin can be any of a wide variety of lignin compositionsfound in nature or as known in the art.

As known in the art, there is no uniform lignin composition found innature. Lignin is a random polymer that shows significant compositionalvariation between plant species. Many other conditions, such asenvironmental conditions, age, and method of processing, influence thelignin composition. Lignins differ mainly in the ratio of three alcoholunits, i.e., p-coumaryl alcohol, guaiacyl alcohol, and sinapyl alcohol.The polymerization of p-coumaryl alcohol, coniferyl alcohol, and sinapylalcohol forms the p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S)components of the lignin polymer, respectively. The precursor lignin canhave any of a wide variety of relative weight percents (wt %) of H, G,and S components. Besides the natural variation of lignins, there can befurther compositional variation based on the manner in which the ligninhas been processed. For example, the precursor lignin can be a Kraftlignin, sulfite lignin (i.e., lignosulfonate), or a sulfur-free lignin.

Lignin is the most abundant aromatic based biopolymer on Earth, but itis chemically recalcitrant to conversion and bioconversion due to theapparent randomness of its chemical composition and physical structure.Lignin can be considered a “glue” or “epoxy” between polysaccharidefibers that provides strength, rigidity, and protection to the cellwalls of vascular plants. From a chemical standpoint, lignin is a highlyheterogeneous polymer formed by the polymerization of phenyl-propanoidmolecules including coniferyl, sinapyl and coumaryl alcohols via aryllinkages, ether linkages, and carbon-carbon bonds.

Based on the assumption that 100 gallons of ethanol are produced from 1ton of biomass and that biomass (e.g., wood and grass) contains onaverage about 20% lignin, one can quickly estimate that a biorefineryoperating on a 100 million gallon per year capacity would produce about200,000 tons of lignin material. To meet a 20% replacement of gasolinefor the U.S. only by 2020, equivalent to about 35 billion gallons ofethanol, a total of approximately 700 million tons of lignin would beproduced per year. The actual production of lignin, mostly Kraft ligninas byproduct of the paper industry, is approximately 90 million tons peryear worldwide. In other words, the lignin production worldwide would beincreased by more than an order of magnitude.

Lignin can be used for low- or high-priced products based on theapplication and the degree of chemical purity. Until recently, marketsfor lignin products have not been large, competitive, or attractiveenough to compensate for the cost of isolation and purification comparedto the recovered energy derived from its burning. This is mainly becausethe cost of oil is still low enough and the supplies are high enough toprovide the building blocks for the chemical and material industries.However, in a carbohydrate economy framework based on biofuels andbioproducts co-production, high-purity isolated lignin dedicated forconversion could be estimated at $1.10 per kg of raw material comparedto $0.04, when used for co-firing. Low-end applications are mostlydirected to dispersants, soil conditioners for carbon sequestration,adsorbents for fertilizers and pesticides, as well as fuels, whichrequire little or no further conversion after extraction. High-endapplications requiring depolymerization of lignin include the productionof phenolic precursors (e.g., DMSO, vanillin, phenol, and aromaticcompounds) and polymer components (e.g., epoxy resins, polyurethanefoams, phenolic resins powders, carbon fibers and glue and binders).

In nature, the conversion of lignin is performed by specialist microbes,particularly fungi and bacteria. Lignocellulosic bacteria and fungushave the ability to depolymerize lignin in order to gain access tocellulosic fractions of biomass. To that end, lignocellulosic bacteriaand fungus excrete an array of oxidoreductase enzymes, which includelaccases, oxidases, and peroxidases, along with organic acids andH₂O₂-producing catalases. The most potent oxidoreductase enzymes areproduced by a specific group of fungi known as white rot fungi, whichspecialize in lignocellulosic degradation. Various types of fungalperoxidases differ in the nature of their substrates.

Lignin peroxidase (LiP, E.C. 1.11.1.14) catalyzes the oxidative cleavageof C—C bonds in a number of model compounds, and oxidizes benzylalcohols to aldehydes or ketones. Typical reactions catalyzed by ligninperoxidases are Cα-Cα cleavage, Cα oxidation, alkyl aryl cleavage,aromatic ring cleavage, demethylation, hydroxylation and polymerization.Lignin peroxidases are involved in the oxidative breakdown of lignin inwhite-rot basidiomycetes. Lignin peroxidase catalyzes the oxidation ofnon-phenolic aromatic rings into aryl cation radicals by H₂O₂. A typicalexample is the oxidation of veratryl alcohol (3,4-dimethoxybenzylalcohol) into veratryl aldehyde (3,4-dimethoxybenz aldehyde) via theintermediary formation of veratryl cation and benzyl radicals: veratrylalcohol+H₂O₂→veratryl aldehyde+2 H₂O. Manganese peroxidase (MnP; E.C.1.11.1.13) has lower redox potentials (up to 1.1 V) than LiP (up to 1.5V) and catalyzes the Mn-mediated oxidation of lignin and phenoliccompounds. This enzyme catalyzes the oxidation of Mn(II) to Mn(III) byH₂O₂. The highly reactive Mn(III) is stabilized via chelation in thepresence of dicarboxylic acid: 2Mn(II)+2H⁺+H₂O₂→2Mn(III)+2H₂O. Thepurpose of MnP is to generate small and potent oxidizing agents thatdiffuse into the lignified cell wall and achieve depolymerization oflignin from within. Versatile peroxidase (syn. hybrid peroxidase,manganese-lignin peroxidase: VeP EC 1.11.1.16) is a fairly newligninolytic enzyme, combining catalytic properties of manganeseperoxidase (oxidation of Mn(II)), lignin peroxidase (Mn-independentoxidation of non-phenolic aromatic compounds) and plant peroxidase(oxidation of hydroquinones and substituted phenols). Any one or acombination of the above-mentioned peroxidases may be used in the lignindepolymerization process described herein.

In a first embodiment, the lignin-containing material is a form oflignin partially or substantially separated from other components ofwood (e.g., cellulosic and hemicellulosic components), as is generallyprovided from a pretreatment process of lignocellulosic material, thedetails of which are well known in the art of lignocellulosic processingand conversion. The pretreatment process serves to either separatelignin from other components of the lignin-containing source, or toweaken the bonds between lignin and the other components. As is alsowell known in the art, the lignin may be further isolated by, forexample, extraction. In a second embodiment, the lignin-containingmaterial is a lignin-containing consumable product, such as paper orcardboard, which may or may not be pretreated. In a third embodiment,the lignin-containing material is a lignin-containing natural source(i.e., raw lignocellulosic material), such as woodchips, grasses (e.g.,switchgrass and mixed grasses), corn stover (e.g., leaves, husks,stalks, or cobs of corn plants), sugarcane, saw dust, hemp, or acombination thereof, all of which are generally pretreated to make thelignin sufficiently available for depolymerization.

In the lignin depolymerization process, any of the BNC or BNC-scaffoldstructures, described above, is contacted with a lignin-containingmaterial under conditions where partial or complete depolymerization oflignin occurs by free-radical activity of the enzyme-bound magneticnanoparticles or aggregates thereof. The BNC or BNC-scaffold structureand the lignin-containing material are generally made to contact bycombining them in an aqueous solution, such as an aqueous solution usedin a pretreatment process of the lignin-containing material. In someembodiments, a room temperature condition (e.g., at least 10, 15, 18,20, or 22° C. and up to 25° C., 30° C., or 40° C., or any range therein)is used during the depolymerization process. In other embodiments, anelevated temperature condition (e.g., above 40° C., or at least or above45, 50, or 60° C., or up to the temperature that the FRP enzyme degradesor suffers a substantial loss in activity) is used during thedepolymerization process. In other embodiments, a reduced temperaturecondition (e.g., below 15° C., or up to or below 10, 5, or 0° C.) isused during the depolymerization process. By being depolymerized, thelignin is broken down into shorter segments compared to its originalform. A complete depolymerization results in the conversion of all or asubstantial portion (e.g., at least 80, 90, or 95%) of the lignin intoat least one or more of the basic building blocks of lignin, i.e.,coniferyl, sinapyl, and coumaryl alcohols, and derivatives thereof. Apartial depolymerization generally results in less than 80%, or up to70, 60, 50, 40, 30, 20, 10, 5, or 1% of lignin being converted toprimary building blocks, with the rest of the lignin being converted tosegments containing two, three, four, or a higher multiplicity (even upto 10, 20, 50, 100, 200, 500, or 1000) of building blocks (e.g.,p-hydroxyphenyl, guaiacyl, and syringyl units derived from coumaryl,coniferyl, and sinapyl alcohols, respectively). Since different degreesof lignin depolymerization may be preferred for different applications,the depolymerization conditions can be suitably adjusted to provide anappropriate degree of depolymerization or to favor one or more types ofdepolymerization products over others.

Since each lignin-containing material has a different distribution andrelative amount of each building block, the relative amount of eachproduct produced from depolymerization is very much dependent on thetype of lignin-containing material. Other depolymerization products,e.g., aromatic aldehydes, ketones, alcohols, and acids, are generallyalso produced during the polymerization process, typically in lesseramounts. In embodiments where such other products are not desired, theymay be advantageously minimized or eliminated as a product by adjustmentof reaction conditions, including appropriate selection of the FRP-boundmagnetic nanoparticle or aggregate thereof.

Any of the BNC or BNC-scaffold structures described above can be usedfor the lignin depolymerization process. In particular embodiments, theenzyme used in the lignin depolymerization process is a FRP, such as aperoxidase, and particularly, a lignin-degrading peroxidase, such as alignin peroxidase, versatile peroxidase, manganese peroxidase, orcombination thereof (including a core-shell combination thereof). TheFRP enzyme may also more particularly be a fungal, microbial, or plantperoxidase. In specific embodiments, the FRP enzyme is a system of twoFRP enzymes, such as a peroxidase combined with a glucose oxidase, or aperoxidase and/or oxidase combined with a laccase.

In some embodiments, the lignin depolymerization process is coupled(i.e., integrated) with a downstream process in which depolymerizationproduct produced in the lignin depolymerization process is used for theproduction of other products. The downstream process may convert lignindepolymerization product into, for example, biofuel or an industrialchemical product, e.g., a polymer, plastic, polymer precursor (monomer),solvent, adhesive, paint, detergent, lubricant, food product, medicinalproduct, or aroma, or a precursor therefore. The downstream process mayalternatively incorporate the lignin depolymerization product into anysuch end product.

In some embodiments, the lignin depolymerization process is coupled withan upstream process in which lignin-containing material is provided foruse in the lignin depolymerization process described herein. Theupstream process can be, for example, a paper or pulp producing process,a biomass-to-biofuel process (i.e., where primarily cellulosic materialis hydrolyzed and converted to biofuel), or a biomass-to-ethanolfermentation process (i.e., where primarily cellulosic material ishydrolyzed and converted to ethanol).

In another aspect, the invention is directed to a process for removingaromatic contaminants from water (i.e., a water remediation process). Inthe process, water contaminated with one or more aromatic substances iscontacted with any of the BNC or BNC-scaffold structures, describedabove, to cause the aromatic substances to precipitate, i.e., asinsoluble material. The precipitated (i.e., sedimented) material ispreferably then further separated, such as by centrifugation orsettling, and removed from the water by, for example, filtration ordecanting. Without being bound by any theory, it is believed that thearomatic substances react with free radicals produced by theenzyme-bound magnetic nanoparticles to produce a polymerized materialderived from the aromatic substances. The aromatic contaminant can beany aromatic substance, including those more commonly found incontaminated water. In some embodiments, the aromatic contaminant isbenzene, or a benzene derivative, such as a halogenated benzene (e.g.,chlorobenzene, dichlorobenzenes, bromobenzenes, or a polychlorinatedbiphenyl, i.e., PCB), alkylbenzene (e.g., toluene, ethylbenzene, or axylene), phenolic substance (e.g., phenol, resorcinol, catechol, apolyphenol, or a substituted phenol, such as cresol), etherified benzene(e.g., anisole), fused ring compound (e.g., naphthalene, or polyaromatichydrocarbon), aromatic amine (e.g., aniline and N-alkyl or N,N-dialkylsubstituted anilines), benzoic acid compound (e.g., benzoic acid, estersthereof, and hydroxy-substituted derivatives of benzoic acid), orbioactive aromatic compound (e.g., as produced by bacteria, fungi, orplants). In other embodiments, the aromatic contaminant is aheteroaromatic substance, such as furan, pyran, dioxin, thiophene,pyridine, pyrazine, pyrimidine, pyrrole, imidazole, indole, andderivatives thereof.

Any of the BNC or BNC-scaffold structures described above can be usedfor the water remediation process. In particular embodiments, the enzymeused in the water remediation process is a FRP enzyme, such ashorseradish peroxidase, or horseradish peroxidase in combination with anoxidase.

In another aspect, the invention is directed to a process forpolymerizing monomers polymerizable by a free-radical mechanism. In theprocess, one or more types of monomers are reacted with any of the BNCor BNC-scaffold structures, described above, to cause the monomers topolymerize. The monomers can be, for example, any of the substancesprovided above for the water remediation process. In particularembodiments, the monomers are or include vinyl-addition monomers. Uponpolymerization, a vinyl-addition polymer is produced. Some examples ofsuch monomers include ethylene, propylene, butadiene, the acrylates andesters thereof, methacrylates and esters thereof, acrylonitriles, vinylacetate, styrene, divinylbenzene, vinyl fluorides, and vinyl chlorides.In other embodiments, the monomers are phenolic compounds. Uponpolymerization, a phenolic resin or polymer is produced. Thepolymerization process can utilize any of the conditions and apparatuseswell known in the art for practicing polymerization reactions, and inparticular, free-radical initiated polymerization reactions.

In another aspect, the invention is directed to a process for theepoxidation of alkenes. In the process, alkenes in the presence ofoxygen are reacted with any of the BNC or BNC-scaffold structures,described above, provided that the BNC or BNC-scaffold structureincludes an oxygen-transfer enzyme, such as a chloroperoxidase or lipaseenzyme. The reaction is advantageously conducted at a significantlylower temperature (e.g., room temperature, ca. 25° C.) compared totemperatures conventionally used in the art for epoxidizing alkenes. Thealkene can be, for example, ethylene or propylene, and the end productcan be, for example, ethylene oxide or propylene oxide.

In particular embodiments, the epoxidation process employs immobilizedchloroperoxidase or lipase, or both, in BNCs immobilized onto magneticscaffolds. The enzyme-containing catalysts may be used with magneticreactors (i.e., by magnet trap methods, as further described below)either in a continuous flow or batch system. In continuous flow systems,the catalysts are retained in the reactors by the magnetic field of theelectromagnets. The reagents (alkene and oxidant) are introducedupstream and react with the enzymes in the reaction zone. The oxidantcan be, for example, hydrogen peroxide or glucose if thechloroperoxidase is used with a glucose oxidase. The solution can beaqueous or organic (such as dioxane) or any other solvent compatiblewith the enzyme. The continuous flow removes the products of thereaction from the vicinity of the enzymes and prevents the accumulationof inhibitory levels of substrates and products. The products aretypically concentrated downstream of the conversion process, due todifference in solubility, and subsequently reacted in a secondaryreaction due to the poor stability of epoxide groups. In batch systems,the reagents are generally mixed with the catalysts, stirred, and thecatalysts removed from the batch by magnetic capture when the reactionis complete. The products of the reactions are concentrated due to adifference in solubility, and then subsequently reacted. The capturedcatalysts can be reused for a new batch reaction.

In another aspect, the invention is directed to a process for thehalogenation of phenols. In the process, phenols are reacted with any ofthe BNC or BNC-scaffold structures, described above, provided that theBNC or BNC-scaffold structure includes a halogenating enzyme, such as achloroperoxidase. The phenol reactant can be phenol itself, or anysuitable phenol derivative, such as any of the phenolic compoundsprovided above. The phenolic product can be, for example, a chlorinated,brominated, or iodated phenol compound.

In particular embodiments, the halogenation process employs immobilizedchloroperoxidase in BNCs immobilized onto magnetic scaffolds. Theenzyme-containing catalysts may be used with magnetic reactors (i.e., bymagnet trap methods, as further described below) either in a continuousflow or batch system. In continuous flow systems, the catalysts areretained in the reactors by the magnetic field of the electromagnets.The reagents (phenol or phenol derivatives, oxidant, and halide ions,such as I⁻, Br⁻ or Cl⁻) are introduced upstream and react with theenzymes in the reaction zone. The oxidant can be, for example, hydrogenperoxide or glucose if the chloroperoxidase is used with a glucoseoxidase. The solution can be aqueous or organic (such as dioxane) or anyother solvent compatible with the enzyme. The continuous flow removesthe products of the reaction from the vicinity of the enzymes andprevents the accumulation of inhibitory levels of substrates andproducts. The products are typically concentrated downstream of theconversion process due to the difference in volatility and solubility.In batch systems, the reagents are generally mixed with the catalysts,stirred, and the catalysts removed from the batch by magnetic capturewhen the reaction is complete. The products of the reactions areconcentrated due to a difference in volatility and solubility. Thecaptured catalysts can be reused for a new batch reaction.

In another aspect, the invention is directed to a process for inhibitinggrowth and function of microorganisms in a solution. In the process,water containing microorganisms (i.e., microbial-contaminated water) istreated with any of the BNC or BNC-scaffold structures, described above,provided that the BNC or BNC-scaffold structure includes an FRP enzyme,such as a peroxidase, or more specifically, a lactoperoxidase (LPO) or alactoperoxidase combined with a glucose oxidase.

The LPO-system is considered to be one of the body's natural defensemechanisms against microbial infections since LPO exhibits broadantifungal and antibacterial activity in the presence of thiocyanate andhydrogen peroxide. Consequently, applications of lactoperoxidase arebeing found in preserving food, cosmetics, and ophthalmic solutions.Furthermore, lactoperoxidase have found applications in dental and woundtreatment. Lactoperoxidase may also find application as anti-tumor andantiviral agents. Lactoperoxidase substrates include bromide, iodide andthiocyanate. The oxidized products produced through the action of thisenzyme have potent bactericidal activities. The lactoperoxidasecatalysts can be used in combination with magnetic trap reactors,described below, to produce the broadly acting hypothiocyanite,hypobromite, and hypoiodite ions from thiocyanate, bromide, and ioditerespectively. These ions are then released with the effluent where theyinduce oxidative stress to the microorganisms downstream, therebydecontaminating the effluent. Alternatively, the catalysts can be usedin a batch reactor or onto a surface in an aqueous solution and thenrecaptured by a magnetized collector. The action of hypothiocyanate, forexample, against bacteria is reported to be caused by sulfhydryl (SH)oxidation. The oxidation of —SH groups in the bacterial cytoplasmicmembrane results in loss of the ability to transport glucose and also inleaking of potassium ions, amino acids, and peptide, thereby inducingthe death of the microorganisms. The products of lactoperoxidase aregenerally considered safe and non-mutagenic, and hence compatible withfood and health applications.

In another aspect, the invention is directed to a process for convertingcarbon dioxide to methanol. In the process, carbon dioxide is reactedwith any of the BNC or BNC-scaffold structures, described above,provided that the BNC or BNC-scaffold structure includes a dehydrogenasesystem containing at least two, three, or at least four dehydrogenaseenzymes. The dehydrogenase enzyme system can include, for example, aformate dehydrogenase (NAD⁺ oxidoreductase, such as EC 1.2.1.2; orferricytochrome-b1 oxidoreductase, such as EC 1.2.2.1), combined with aformaldehyde dehydrogenase (e.g., EC 1.2.1.2) or a cytochrome informate:ferricytochrome-b1 oxidoreductase (e.g., EC 1.2.2.1); an alcoholdehydrogenase (EC 1.1.1.1); and glucose dehydrogenase (EC 1.1.99.10).

The carbon dioxide is converted to formic acid by the formatedehydrogenase, the formic acid is converted to formaldehyde by theformaldehyde dehydrogenase, and the formaldehyde is converted tomethanol by an alcohol dehydrogenase. A glucose dehydrogenase isrecycling the NAD⁺ cofactors from the formate dehydrogenase,formaldehyde dehydrogenase, and alcohol dehydrogenase reaction withtheir substrates (CO₂, formic acid, and/or formaldehyde) and NADH. Thetheoretical molar ratio is three molecules of glucose to convert onemolecule of CO₂ to methanol. In particular embodiments, the formatedehydrogenase, formaldehyde dehydrogenase, and alcohol dehydrogenase areentrapped individually or together inside the BNCs. The BNCs are thentemplated onto the magnetic scaffold. The BNCs made with the glucosedehydrogenase are then added to the previous catalysts. Thisconfiguration permits the trapping and recycling of cofactors andmaximizes their use at the vicinity of the formate dehydrogenase,formaldehyde dehydrogenase, and alcohol dehydrogenase.

Alternatively, the CO₂ conversion process can be decoupled with threedistinct sequential reactions using the formate dehydrogenase andglucose dehydrogenase catalysts for the synthesis of formic acid fromCO₂, then formaldehyde dehydrogenase and glucose dehydrogenase catalystsfor the synthesis of formaldehyde from formic acid, then alcoholdehydrogenase and glucose dehydrogenase for the synthesis of methanolfrom formaldehyde. Separate reaction zones in flow reactors or separatebatch processes can be used. Hence, the process can be used to produceformic acid, formaldehyde, and/or methanol.

For any of the processes described above, the BNC-scaffold structurescan advantageously be captured by magnetic separation in order toprevent contamination of the final product. Moreover, a furtheradvantage of the BNC-scaffold structures described herein is theirability in many cases to retain their activity and re-form aftercapture, which permits them to be re-used after capture, therebyincreasing the total turnover number (TTN) of the enzymes. BNC-scaffoldsystems showing a loss of activity after several cycles canadvantageously be easily extracted and concentrated to their solid formto provide a less wasteful and more efficient process. In particular,metal-coated BNCs can be repurposed by denaturation of the enzymes,sonication, and purification in order to be restored and re-used withfresh functional enzymes. BNC-scaffold structures are attractive forprocess applications that use lower intensity magnetic fields. TheBNC-scaffold structures maintain stable, nanosized, and mesoporousstructures, which helps to maintain enzyme activity while increasing theoverall density and mass susceptibility of the magnetic catalyst. Theseultrastructures lend themselves to easier manipulation by externalmagnetic fields as produced by permanent small magnets and weak fieldelectromagnets. The reaction solution can be purged and replaced whilethe BNC-scaffold structures are magnetically trapped, hence allowing forsequential use of the BNC-scaffold structures as long as the enzymeretains process level activities.

In yet another aspect, the invention is directed to a magnetic trapmethod for increasing a space time yield and/or total turnover number ofa liquid-phase chemical reaction that includes magnetic particles tofacilitate the chemical reaction. In the method, a liquid-phase chemicalreaction that includes any of the BNCs or BNC-scaffold structuresdescribed above is subjected to a plurality of magnetic fields (i.e.,“dynamic magnetic trap reactors” or “DMTRs”) by one or a plurality ofelectromagnets, each of which may be independently adjusted to provide amagnetic field of desired magnetic strength, relative position in theliquid-phase chemical reaction, and relative motion to spatially confinethe magnetic particles. In the method, the magnetic strength, relativepositioning, and relative motion of the plurality of magnetic fields areprovided by a system of electromagnets in which current flow isappropriately controlled or adjusted. In particular, the space timeyield can be increased by applying the magnetic fields in a manner thatconfines the reaction volume space.

Any of the BNCs or hierarchical catalyst assemblies thereof, describedabove, can be used in the magnet trap method. In particular embodiments,the BNCs or hierarchical catalyst assemblies thereof advantageouslybehave in a “fluidic” manner during the course of the reaction whileunder the influence of the moving external magnetic fields. The fluidicmotion can be characterized by a congregation (i.e., “cloud”) of theBNCs or hierarchical catalyst assemblies collapsing at impact with thewall of the reactor when attracted by the electromagnets. By doing so,the products of the reaction of the enzymes are expelled from thecatalysts. When the “cloud” is moving in the other direction, it absorbsfresh substrate, which reacts with the enzymes, and then the productsare expelled again when the “cloud” hits the opposite wall. This is asignificant feature of the magnetic scaffolds since this behavior canpermit them to function as a “micropump” (i.e., similar to squeezing asponge).

In some embodiments, the confinement results in at least a first andsecond reaction zone in which separate reactions can be conducted ineach reaction zone. The purpose of the foregoing embodiments is to avoidthe accumulation of substrates and products in the vicinity of thecatalysts. The free radicals generated by the enzymes are highlyreactive and can react with the enzymes or polymerize at the surfaces ofthe catalysts. To avoid such conditions that would be detrimental to theoverall process and efficiency of the enzymes, the process is decoupledin a reaction volume 1 where the free radicals are generated, and areaction volume 2, void of catalyst, where they react with each other.The magnetic catalysts are maintained in the reaction volume 1 by thealternating magnetic field generated by the electromagnets while thesolution and the reagents flow through the catalysts, react, and arecarried away by the flow. The parameters controlling the process areprimarily the intensity of the induced magnetic field and the frequencyof the bouncing motion that confine the magnetic catalysts and the flowrate of the solution in the reactor.

In other embodiments, the magnetic trap method includes magnetic captureof the magnetic particles by using the dynamic magnetic trap reactorsafter the reaction has reached completion. In this configuration, theelectromagnets are turned on in order to capture all of the magneticcatalyst. The solution containing the products of the reaction istypically removed or flushed out for further processing. The batch isgenerally replenished with fresh solution with the substrates to beconverted. Typically, the electromagnet arrays resume the cycling poweron/power off to agitate the magnetic catalysts in the batch. When thecatalysts are reaching their end-usage, they can be captured by theelectromagnets, and the reactor can be filled up with rinsing solution.The electromagnets can then be turned off to free the magnetic catalyststhat are flushed out with the rinsing solution. The magnetic catalystscan be concentrated and extracted from the rinsing solution with asecondary array of electromagnets in a downstream secondary process.

In particular embodiments of the magnetic trap method, current flow tothe electromagnets is controlled by a computer program to provide adesired set of magnetic strengths, relative positions in theliquid-phase chemical reaction, and/or relative motions of the pluralityof magnetic fields that cause spatial confinement of the magneticparticles. Any desirable number of electromagnets may be used, and theelectromagnets can be positioned in any suitable manner around thereaction vessel to achieve, for example, reaction volume confinement,separation of reactions, or magnetic capture of the magnetic particles.The electromagnets may also be arranged in arrays, such as two arrays oftwo electromagnets, two arrays of three electromagnets, two arrays offour electromagnets, three arrays of two electromagnets, three arrays ofthree electromagnets, and so on, with each individual electromagnet oreach array of electromagnets independently operated and controlled.

For example, a single-helix or double-helix arrangement of theelectromagnets may be used, as particularly depicted in FIGS. 2A and 2B,respectively. The magnetic arrays include computer-controlled andprogrammable electromagnets that maintain the magnetic catalysts in thereaction zone and prevent the leaching of the enzymes with the solution.As shown in FIGS. 2A and 2B, as particularly applied to thepolymerization of aromatics or remediation of water containing sucharomatics, the array of electromagnets can be appropriately operated tomagnetically trap the BNCs or BNC-scaffold assemblies to promote freeradical polymerization in a defined section or plurality of sections ofthe reaction vessel, while other parts of the reaction volume areavailable for non-magnetic induced reactions or physical processes, suchas polymer condensation or sedimentation. The separation of theoxidation of the aromatics and the polymerization thereof prevent theformation of the polyphenols close to the catalysts. The molecularentrapment of the catalysts (and enzymes) with polyphenol is referred toas product inhibition. Product inhibition by molecular entrapment isirreversible and translates to drastic loss of catalytic efficiency.Moreover, in the separated reactors, polymerization and condensation canbe favored in the bottom part of the reactor by inclusion of coagulatingagents, such as salts or divalent cations, or by inclusion of couplingsurfaces, such as sand or organic materials, that trap the polyphenolsand free radicals.

The motion of magnetic fields can be any suitable motion, dependent onthe desired result, such as scanning vertical (up and/or down) motion,scanning horizontal (left and/or right) motion (e.g., “ping-pongmotion”), figure eight (“8”) motion, diagonal up and down (“V”) motion,double diagonal (W motion), scanning horizontal, diagonal down scanninghorizontal (Z motion). Other types of motion may be desirable to controlthe motion of the catalysts in solution provided that the flow of liquidis compensated by the counter force generated by the electromagnets. Themotion is the result of the geometry of the electromagnets related tothe geometry of the reactor and the cycling of the power on/off of theelectromagnets. The speed of the catalyst motion is determined by thefrequency of the electromagnets (power on/power off) and the strength ofthe magnetic field from these magnets, which is a function of thecurrent intensity provided to the electromagnets. A computer program canbe devised to make any desired motion.

For example, a computer program can be devised to provide an alternatingor “ping-pong” motion of the magnetic field. The ping-pong motion isachieved by sequentially turning on one magnet and shutting down itscounterpart. An example of such a program for inducing a ping-pongmotion is as follows:

int period=1000; //Set the period in msec void setup( ){ pinMode(6,OUTPUT); //Set the u-axis output pin pinMode(6, OUTPUT); paired with 9pinMode(7, OUTPUT); //Set the u-axis output pin; paired with 10pinMode(8, OUTPUT); //Set the u-axis output pin; paired with 11pinMode(9, OUTPUT); //Set the v-axis output pin ; paired with 6pinMode(10, OUTPUT); //Set the v-axis output pin ; paired with 7pinMode(11, OUTPUT); //Set the v-axis output pin ; paired with 8 } voidloop( ){ digitalWrite(6, HIGH); //Switch on u-axis digitalWrite(7, LOW);//Switch off u-axis digitalWrite(8, HIGH); //Switch on u-axisdigitalWrite(9, LOW); //Switch off v-axis digitalWrite(10, HIGH);//Switch on v-axis digitalWrite(11, LOW); //Switch off v-axisdelay(ceil(period/2)); //Delay for the given interval digitalWrite(6,LOW); //Switch on u-axis digitalWrite(7, HIGH); //Switch off u-axisdigitalWrite(8, LOW); //Switch on u-axis digitalWrite(9, HIGH); //Switchoff v-axis digitalWrite(10, LOW); //Switch on v-axis digitalWrite(11,HIGH); //Switch off v-axis delay(ceil(period/2)); //Delay for the giveninterval }

The invention is also directed to an apparatus for conducting themagnetic trap method described above. The apparatus should include atleast a reaction chamber constructed of a suitable reaction vesselmaterial and having at least two opposing walls; one or more (typically,arrays of) electromagnets arranged on external surfaces of the at leasttwo opposing walls; and a computer-programmable controller forcontrolling current flow in the electromagnets. The number ofelectromagnets is scalable based on the controllers being used, or bythe use of multiple controllers. The reaction chamber can be designedas, for example, a batch reactor, or alternatively, a flow cell forcontinuous production. The design and construction of such reactionvessels are well known in the art. In typical embodiments, the apparatusalso includes means for individually controlling the electromagnets byuse of one or more computer-programmable controllers, which control thefrequency of the on/off switch of the electromagnets. Thecomputer-programmable controllers may have the electronic set up shown,for example, in FIG. 3. In the diagram in FIG. 3, the programmableelectronic board can employ, for example, a 12 V power source and 6outputs (+5 V), shown for exemplary purposes only. The microcontrollercontrol inputs (“CtrInp” designations) can be as shown, for example, inFIG. 4, which depicts a typical commercially available controller board(e.g., Arduino™ UNO) that has 14 digital input/output pins (of which sixcan be used as PWM outputs), six analog inputs, a 16 MHz crystaloscillator, a USB connection, a power jack, an ICSP header, and a resetbutton. Each output can control an electromagnet or an array ofelectromagnets. The board is powered by an external power generatorproviding the 12V source. The output of +5V is against the ground(0/+5V). The USB port is used to program the microcontroller board.

Examples Lignin Depolymerization

Peroxidase enzymes have been amply investigated due to the potential oftheir oxidative activity for industrial sectors. Yet, they areparticularly prone to substrate-inhibition, which has preventeddevelopment of large-scale peroxidase-based biotechnologies. Herein isdemonstrated that the activity and resilience of fungal ligninolyticperoxidases can be dramatically increased in association withgold-coated magnetic nanoparticles (Au-MNPs). The assemblies havesuperior activity than the free enzyme systems and can be applied to theenhanced depolymerization of the lignin component of energy feedstocks.The results show that the assemblies can encompass complex enzymesystems to overcome the current physical and biochemical limitations ofthis family of enzymes and create a new generation of lignin catalysts.The enzyme-based catalysts exhibited a bimodal activity with two maximabetween 0.1 and 1 mM and above 500 mM of H₂O₂. The Au-MNPs had noactivity in the range of concentration optima for the enzymes. As shownin FIGS. 5 and 6, differences were observed on the initial rates ofcatalyst made with two types of magnetite nanoparticles, Au-M90 andAu-M60, where Au-M90 had a more pronounced effect on both velocities andoptimal H₂O₂ concentration. M60 refers to nanoparticles of magnetiteformed at 60° C., and M90 refers to magnetite nanoparticles formed at90° C. The temperature during formation affects the crystallite size andultimately the magnetic properties for the nanoparticles. M90nanoparticles are about 10 nm average diameter while M60 nanoparticlesare about 8 nm average diameters.

FIG. 5 is a graph showing the effect of H₂O₂ on DMP oxidation velocitycatalyzed by manganese peroxidase at 2.5 nM, Au-M60 at differentconcentrations, and BNCs thereof at pH 3.5 in malonate buffer (50 mM).The x-axis is on a log₁₀ scale for convenience. The coating with goldprevents the dissolution of the magnetic core (magnetite) in the buffernecessary for enzymes activities. The BNCs (gold coated nanoparticles)increase the activity and lower the inhibition of manganese peroxidase.The inhibition is 50 times less when the enzymes are embedded ingold-coated nanoparticle clusters. The nanoparticles have no intrinsiccatalytic activities in this range of concentration of hydrogen peroxide(0.001 to 10 mM).

FIG. 6 is a graph showing the effect of H₂O₂ on DMP oxidation velocitycatalyzed by versatile peroxidase at 2.5 nM, Au-M60 at differentconcentrations, and BNCs thereof at pH 3.5 in malonate buffer (50 mM).The x-axis is on a log₁₀ scale for convenience. The BNCs (gold coatednanoparticles) increase the activity and lower the inhibition ofversatile peroxidase. The inhibition is 28 times less when the enzymesare embedded in gold-coated nanoparticles. The nanoparticles have nointrinsic catalytic activities in this range of concentration ofhydrogen peroxide (0.001 to 10 mM).

As shown in FIGS. 5 and 6, with increasing concentrations of Au-MNPs,the peak velocities decreased but the optimal concentration of H₂O₂ wasshifted toward higher concentrations. This was reflected in the kineticparameters as the V_(max) decreased with increasing concentration ofMNPs. The inhibition constant (K_(i)) of the enzyme increased withincreasing concentration of Au-MNPs. Compared to free manganeseperoxidase, the inhibition decreased by 50 fold with Au-M90, and about30 times with Au-M60. Remarkably, it is believed to be the lower K_(m)and the higher K_(i) values that drove the relative increase in velocityobserved for BNCs. The coating with gold prevents the dissolution of themagnetic core (magnetite) in the buffer necessary for enzymesactivities. Without gold-coating, magnetite nanoparticles are typicallyfully dissolved in 10 minutes below pH 5 in malonic acid. The BNCs (goldcoated nanoparticles) increase the activity and lower the inhibition ofmanganese peroxidase.

The peroxidase-based catalytic system was applied to thedepolymerization of grass biomass, with the production of solublearomatics followed by UV and fluorescent spectroscopy. For thisapplication, the manganese peroxidase and versatile peroxidase werecombined in equimolar ratio. Initial tests using hydrogen peroxidedirectly were not conclusive for the amount of enzyme used. Theconcentration of peroxide needed to detect any activity of biomass wouldbe inhibitory if added at once. Enzyme stability can be notably improvedby maintaining a lower oxidant concentration over the course of thereaction. In this case, background oxidations are reduced by keeping thehydrogen peroxide concentration to low levels by carefully controllingthe inputs of peroxide. This effect of excess of hydrogen peroxide“shutting down” enzymes is well known and has been classically addressedby complex reactor designs to precisely control the H₂O₂ feeding streamswith feedback loops. To circumvent this issue, the in situ production ofH₂O₂ was chosen by coupling peroxidase enzymes with a peroxide producingenzyme such as the well-known glucose oxidase (Gox). Gox convertsglucose to hydrogen peroxide and gluconolactone in the presence ofoxygen.

The glucose oxidase-peroxidase system was combined to assemble with thegold-coated nanoparticles for biomass conversion. As shown in FIGS. 7A,7B, 8A, 8B, and 8C, the hybrid systems of peroxidase+oxidase have higheractivities on biomass than enzymes alone. FIG. 7A shows the UV-visspectra of the supernatant after treatment with a mixture of manganeseperoxidase and versatile peroxidase immobilized or not withingold-coated BNCs. FIG. 7B shows the same spectra when the referencebiomass spectra has been subtracted. In these experimental conditionsand using hydrogen peroxide as oxidant, the enzymes only have anactivity on the release of aromatic compounds from biomass if they areimmobilized in BNCs. Adding peroxide directly in the biomass slurriescan induce a substrate inhibition of the enzymes. Hence, peroxide needsto be added incrementally in small quantities to avoid reachinginhibitory levels. To circumvent this, the in situ production ofhydrogen peroxide was implemented by adding glucose oxidase (Gox) withinthe peroxidase BNCs (FIG. 8A, 8B, 8C). Gox converts glucose togluconolactone and hydrogen peroxide in presence of oxygen. Thisoxidase/peroxidase system permits adjustment in the rate of productionof hydrogen peroxide by the oxidase to meet the rate of consumption bythe peroxidase. In the appropriate steady-state conditions, the hydrogenperoxide does not reach inhibitory concentration. The catalysts (BNCs)were tested on real biomass and the release of aromatics molecules (fromlignin) was monitored in the UV range (spectra corrected and subtractedagainst control samples). By comparing FIGS. 8A, 8B and 8C, an optimalratio of 1 molecule of Gox for 4 molecules of peroxidase enzymes wasfound to be more appropriate. The activity of the BNCs embeddingmanganese peroxidase, versatile peroxidase, and glucose oxidase wasfound to be about 30% higher on real biomass compared to the freeenzymes system. On real biomass, as shown in FIGS. 8B and 8C, theAu-MNPs protect against inhibition when the concentration of peroxide ismore detrimental to the free enzymes.

The BNCs increase the release of soluble aromatics from biomass whenmanganese peroxidase and versatile peroxidase are used in combinationand in presence of manganese. The BNCs made with gold-coated magneticnanoparticles can be immobilized on magnetic scaffolds for ease ofreusability. The rate of production of hydrogen peroxide can becontrolled by modifying the concentration of glucose oxidase in BNCs.However, gold coated nanoparticles with more gold (e.g., AuM90) providehigher rates of conversion due to lower inhibition of the enzyme fromhigher concentration of hydrogen peroxide.

Phenol Removal

In this example is reported a new family of hierarchical hybridcatalysts containing horseradish peroxidase (HRP) assembled withmagnetic nanoparticles and their incorporation into microparticles, andtheir use in advanced oxidation processes and removal of phenol. Thehierarchical hybrid catalysts can be assembled by the process shown inFIG. 9. The hybrid peroxidase catalysts exhibit activity three timeshigher than free HRP and are able to remove three times more phenolcompared to free HRP under similar conditions. Phenol in this case is amodel molecule representative of phenolic compounds; the efficacy ofBNCs towards phenol removal was evaluated.

As shown in FIG. 10, the templating of the BNCs onto magnetic scaffoldsdoes not change the activities of the BNCs trapped enzymes that are moreefficient than free enzymes. The results show that all systems activelyremove phenol but the BNCs are more effective than the free enzyme forthe same concentration of HRP. Moreover, to reach more than 90% phenolremoval, the amount of HRP required in the BNCs is three to four timesless than that required for free HRP.

The assembly of BNCs onto magnetic scaffold can be controlled by theratio of magnetic microparticles to BNCs as shown in the micrographs inFIGS. 12A and 12B. In FIG. 12A, there is an excess of BNCs that can beseen on the surface of the sample holder surface. In FIG. 12B,additional magnetic scaffold was added to capture the excess BNCs, whichresults in an absence of free BNCs. The foregoing results demonstratethe process of templating the BNCs onto magnetic scaffolds to createhierarchical structures. At least one advantage of such structures isillustrated in the phenol removal results shown in FIGS. 11 A and 11B.Compared to FIG. 11A, templated BNCs (FIG. 11B) can be easily removedand retain activity for several reaction cycles in batch conditions.While the activity of the free enzyme drops quickly after the firstcycle, the templated BNCs retain significant activity after the fifthcycle in these batch conditions compared to the free enzymes.

In general, HRP catalyzes the oxidation of phenolic compounds in thepresence of H₂O₂, thereby producing free radicals. The phenoxy radicalssubsequently react with each other in a non-enzymatic process tocondense or to form polymers. To use the hierarchical catalysts in acontinuous flow fashion, a reactor system composed of electromagnets washerein designed. This system is illustrated in FIG. 13 and FIG. 14.These figures depict, respectively, a V-shape configuration and anI-shape configuration. The back-and-forth motion of the catalysts drivenby the electromagnets maintains the catalysts in a given reaction zoneof the reactors. The motion of the catalysts is driven by the geometryof the electromagnet arrays, sequence of on/off powering of theelectromagnets, and intensity of the magnetic field generated by theelectromagnets. Applied to remediation of phenols and aromatics, theconfiguration permits segregating reaction zones inside the volume ofthe reactors.

In the case of phenol, using a suitable electromagnet configuration,such as described above, the production of the free radicals can beseparated from the polymerization of the polyphenols, as schematized inFIG. 17. The enzymes (and enzyme trapped within BNCs) oxidize the phenolto its free radical form. These highly reactive radicals react with eachother to form polyphenols. These polyphenols can be further condensed byinclusion of a coagulating agent or divalent salt. The phenol removal(polymerization) process described above is generically illustrated inFIG. 15. The brown particles shown in the right portion of the schemeare polyphenols resulting from the polymerization by the BNCs. Thesemicrometric to millimetric size particles of polyphenols can be easilyremoved by sedimentation or filtration.

An example of integration of such reactors for water remediationapplications is illustrated in FIG. 16. The magnetic reactor maintainsthe enzyme-based catalysts against the flow of water and is placedbetween the reservoir of contaminated fluid and the receiving reservoir.The polymerized contaminants are separated from the water by beingpassed through a particulate bed, such as sand or other particulatematerials. The water can be cycled several times in the system accordingto the severity of the contamination.

The hybrid catalysts, as described above, reduce substrate inhibitionand limit inactivation from reaction products, which are common withfree or conventionally immobilized enzymes. Reusability is improved whenthe HRP/magnetic nanoparticle hybrids are supported on micron-sizedmagnetic particles, and can be confined with the specially designedmagnetically-driven reactors described above. The reported performanceof the hybrid catalyst makes them attractive for several industrial andenvironmental applications and pave the way for practical applicationsby eliminating most of the limitations that have prevented the use offree or conventionally immobilized enzymes.

While there have been shown and described what are at present consideredthe preferred embodiments of the invention, those skilled in the art maymake various changes and modifications which remain within the scope ofthe invention defined by the appended claims.

1. A hierarchical catalyst composition comprising a continuousmacroporous scaffold in which is incorporated self-assembled mesoporousaggregates of magnetic nanoparticles, wherein a first enzyme is embeddedin said mesopores of the mesoporous aggregates of magneticnanoparticles, wherein said enzyme functions by converting a diffusiblesubstrate into a diffusible product.
 2. (canceled)
 3. The composition ofclaim 1, wherein said enzyme is comprised of a free-radical producingenzyme.
 4. The composition of claim 3, wherein said enzyme is comprisedof an oxidoreductase.
 5. The composition of claim 4, wherein saidoxidoreductase is comprised of a peroxidase.
 6. The composition of claim5, wherein the peroxidase is selected from horseradish peroxidase,manganese peroxidase, lignin peroxidase, versatile peroxidase,chloroperoxidase, and lactoperoxidase.
 7. The composition of claim 1,further comprising a second enzyme.
 8. The composition of claim 7,wherein said first enzyme is a peroxidase and said second enzyme isglucose oxidase.
 9. The composition of claim 7, wherein said firstenzyme is a peroxidase and said second enzyme is a laccase.
 10. Thecomposition of claim 1, further comprising magnetic particles, notbelonging to said mesoporous aggregates of magnetic nanoparticles,embedded in said continuous macroporous scaffold.
 11. The composition ofclaim 1, wherein said continuous macroporous scaffold has a polymericcomposition.
 12. The composition of claim 1, wherein said continuousmacroporous scaffold has macropores having a pore size of at least about50 nm to about 100 μm. 13.-15. (canceled)
 16. A method for producing ahierarchical catalyst assembly, comprising contacting a plurality ofself-assembled magnetic nanoparticle-enzyme mesoporous aggregates with acontinuous macroporous scaffold in solution to substantially embed saidmagnetic nanoparticle-enzyme mesoporous aggregates into a plurality ofmacropores of said continuous macroporous scaffold.
 17. The method ofclaim 16, wherein said continuous macroporous scaffold is produced by atemplation process comprising (i) producing a composite comprising ascaffold precursor material having a sacrificial templating agentembedded therein, and (ii) selective removal of said sacrificialtemplating agent to produce macropores in said scaffold precursormaterial.
 18. A method for depolymerizing lignin, the method comprisingexposing said lignin to a hierarchical catalyst composition comprising amacroporous scaffold in which is incorporated a plurality ofself-assembled mesoporous aggregates of magnetic nanoparticlescomprising a free-radical-producing enzyme embedded in said mesopores,exposing said mesoporous aggregates to hydrogen peroxide, allowing saidfree-radical-producing enzyme to produce free-radicals from saidhydrogen peroxide that diffuse from said mesoporous aggregates, anddepolymerizing said lignin with said free radicals.
 19. The method ofclaim 18, wherein said depolymerized products are selected from thegroup consisting of coniferyl, sinapyl, and coumaryl alcohols, andderivatives thereof.
 20. A method for removing aromatic substances fromwater, the method comprising contacting water containing said aromaticsubstances with a hierarchical catalyst composition comprising acontinuous macroporous scaffold in which is incorporated a plurality ofself-assembled mesoporous aggregates of magnetic nanoparticles, whereina free-radical-producing enzyme is embedded in said mesopores of themesoporous aggregates of magnetic nanoparticles that polymerizes saidaromatic substances, and removing said polymerized aromatic substancesfrom said water.
 21. The method of claim 20, wherein said aromaticsubstances are selected from substituted phenols, polyphenols, aromaticamines, bioactive aromatics, and mixtures thereof.
 22. A method forpolymerizing monomers polymerizable by a free-radical reaction, themethod comprising reacting said monomers with a hierarchical catalystcomposition comprising a continuous macroporous scaffold in which isincorporated a plurality of self-assembled mesoporous aggregates ofmagnetic nanoparticles, wherein a free-radical-producing enzyme isembedded in said mesopores of the mesoporous aggregates of magneticnanoparticles that causes said polymerization.
 23. A method forepoxidation of alkenes, the method comprising reacting alkenes in thepresence of oxygen with a hierarchical catalyst composition comprising acontinuous macroporous scaffold in which is incorporated a plurality ofself assembled mesoporous aggregates of magnetic nanoparticles, whereinan oxygen-transfer enzyme is embedded in said mesopores of themesoporous aggregates of magnetic nanoparticles that produces an alkeneoxide.
 24. The method of claim 23, wherein said oxygen-transfer enzymeis a chloroperoxidase or a lipase.
 25. A method for halogenationreactions of phenols, the method comprising reacting a phenol with ahierarchical catalyst composition comprising a continuous macroporousscaffold in which is incorporated a plurality of self-assembledmesoporous aggregates of magnetic nanoparticles, wherein a halogenatingenzyme is embedded in said mesopores of the mesoporous aggregates ofmagnetic nanoparticles that produces a halogenated phenol.
 26. Themethod of claim 25, wherein said halogenating enzyme is achloroperoxidase.
 27. A method for inhibiting the growth ofmicroorganisms in a solution, the method comprising exposing saidsolution with a hierarchical catalyst composition comprising acontinuous macroporous scaffold in which is incorporated a plurality ofself-assembled mesoporous aggregates of magnetic nanoparticles,comprising a free-radical-producing enzyme embedded in said mesopores,exposing said mesoporous aggregates to hydrogen peroxide, allowing saidfree-radical-producing enzyme to produce free-radicals from saidhydrogen peroxide that diffuse from said mesoporous aggregates, andinhibiting the growth of said microorganisms with said free-radicals.28. The method of claim 27, wherein said free-radical-producing enzymecomprises a lactoperoxidase.
 29. The method of claim 28, wherein saidlactoperoxidase is combined with a glucose oxidase.
 30. A method forcarbon dioxide conversion to methanol, the method comprising reactingcarbon dioxide with a hierarchical catalyst composition comprising amacroporous scaffold having a plurality of self-assembled mesoporousaggregates of magnetic nanoparticles comprising a formate dehydrogenase,a formaldehyde dehydrogenase, an alcohol dehydrogenase, and a glucosedehydrogenase embedded in said mesopores of the mesoporous aggregates ofmagnetic nanoparticles that produces methanol from carbon dioxide.31.-35. (canceled)
 36. An apparatus for increasing the space time yieldof a liquid-phase chemical reaction containing a plurality of magneticparticles, the apparatus comprising a reaction chamber, a plurality ofspacially opposed electromagnets positioned on an external surface ofsaid reaction chamber, and a controller for controlling a current flowin one or more of said electromagnets, wherein said electromagnets areoperable to control the location of said magnetic particles within saidreaction chamber at a plurality of time points.
 37. (canceled)
 38. Theapparatus of claim 36, wherein said reaction chamber is a batch reactor.39. The apparatus of claim 36, wherein said reaction chamber is a flowcell reactor.